EP3494127B1 - Baculovirus expressionssystem - Google Patents

Baculovirus expressionssystem Download PDF

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EP3494127B1
EP3494127B1 EP17758598.1A EP17758598A EP3494127B1 EP 3494127 B1 EP3494127 B1 EP 3494127B1 EP 17758598 A EP17758598 A EP 17758598A EP 3494127 B1 EP3494127 B1 EP 3494127B1
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gene
replication
baculovirus
essential
genes
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EP3494127A1 (de
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Martine Cerutti
Sylvie JULIANT
Sylvie THERY
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Centre National de la Recherche Scientifique CNRS
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
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Centre National de la Recherche Scientifique CNRS
Institut National de Recherche pour lAgriculture lAlimentation et lEnvironnement
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    • C—CHEMISTRY; METALLURGY
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14151—Methods of production or purification of viral material

Definitions

  • the invention relates to a method for preparing a recombinant baculovirus comprising at least two exogenous genes, sets of homologous recombination implemented in this method of preparation, the recombinant baculoviruses obtained by this method, as well as the use of the recombinant baculoviruses for the production of polypeptides.
  • Baculoviruses are a family of rod-shaped viruses, specific to arthropods, which has four genera (Alphabaculovirus, Betabaculovirus, Deltabaculovirus, Gammabaculovirus) comprising 49 species. Baculoviruses are not able to replicate in cells of mammals or other vertebrates.
  • the baculovirus genome consists of a double-stranded, circular DNA molecule, between 80 and 180 kbp in size.
  • the baculovirus genome associates with highly basic proteins of 6.5 kDa, within a helically symmetrical nucleocapsid, which contains a capsid protein of 39 kDa.
  • the size of the genome determines the length of the nucleocapsid.
  • the nucleocapsid is then enclosed in a lipoprotein envelope to form the viral particle or virion.
  • These structures can be covered with a crystalline or polyhedral matrix consisting essentially of a single protein (polyhedrin) of about 30 kDa.
  • Polyhedra are large structures ranging in size from 1 to 15 ⁇ m in diameter with a polysaccharide outer shell that provides additional protection.
  • Baculoviruses whose genome has been genetically modified, are used in biotechnology for the production of recombinant proteins (i.e. recombinant baculoviruses). After entering an insect cell, these recombinant baculoviruses will use the machinery of the insect cell to produce the recombinant protein.
  • Recombinant baculoviruses are obtained by inserting one or more genes from other species (eg humans, other vertebrates, plants, bacteria and viruses) into the genome of a parent baculovirus. These genes are placed under the control of a viral or cellular promoter (for example the polyhedrin gene promoter) to generate a recombinant baculovirus.
  • the promoter allows transcription of the foreign gene into messenger RNA which in turn is translated into protein in the cell of an insect infected with the recombinant baculovirus.
  • the advantage of using this system is that the level of production of the recombinant protein in the insect cells infected with the recombinant baculovirus can be very high.
  • the recombinant protein can then be purified from the infected cells if the protein is intracellular or from the culture medium if the protein is secreted.
  • the baculovirus expression system is widely used in industry and in research laboratories. In addition to the high productivity of the baculovirus expression system, this system is also highly appreciated because it makes it possible to produce biologically active recombinant proteins. Indeed, insect cells generally make it possible to obtain appropriate post-translational modifications.
  • the baculovirus expression system is difficult to implement on an industrial scale for the production of several distinct recombinant polypeptide sequences, for example proteins comprising several distinct subunits, such as antibodies or couples viral proteins.
  • the methods implemented for the manufacture of recombinant baculoviruses are complex and require a large number of steps which do not make it possible to obtain homogeneous and stable recombinant baculovirus genomes.
  • the homogeneity and stability of recombinant baculovirus genomes are necessary criteria for considering industrial development.
  • the applicant has developed a particularly effective and easy-to-implement process for preparing homogeneous and stable recombinant baculoviruses, and which makes it possible to envisage development at the industrial level, in particular for the production of several distinct recombinant polypeptide sequences, in particular proteins comprising several distinct subunits.
  • the present description relates to a recombinant baculovirus comprising (ie “recombinant baculovirus whose genome comprises” or “recombinant baculovirus whose genome encodes”) n nucleotide sequences of formula (I): [exogenous gene]-[intercalary nucleotide sequence]-[gene essential for functional viral replication] (I), said spacer nucleic acid sequence consists of 0 (zero) to 600 bp, said functional viral replication essential gene is selected from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef- 11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF
  • the present description relates to a cell comprising a recombinant baculovirus of the invention or a set of homologous recombination elements of the invention.
  • the present description relates to the use of a recombinant baculovirus of the invention or of a cell of the invention for the production of n exogenous polypeptides encoded by n exogenous genes.
  • baculovirus genome is intended to designate all the genetic material of a baculovirus, comprising in particular all the coding sequences and non-coding DNA of a baculovirus.
  • replication-deficient baculovirus genome is intended to designate a baculovirus genome in which n genes essential for viral replication have been either deleted (entirely or partially) or mutated from such that the baculovirus genome has lost its ability to replicate in an insect cell.
  • the gene essential for viral replication is no longer expressed or it is transcribed and then translated into a non-functional protein.
  • the deleted (entirely or partially) or mutated genes are called “non-functional genes essential for viral replication”.
  • the baculovirus genomes deficient for viral replication are produced from parent baculovirus genomes using molecular biology techniques well known to those skilled in the art, and in particular allowing the insertion and/or deletion of nucleotide sequences in the parent baculovirus.
  • the baculovirus genome deficient for viral replication comprises at least one nucleotide sequence which allows it to replicate within a bacterial cell. These nucleotide sequences are not exogenous genes within the meaning of the invention.
  • An example of a bacterial replication element is the "Mini-F" nucleotide sequence. Such replication elements are well known in the state of the art.
  • the bacterial cell may be Escherichia coli.
  • a baculovirus genome which comprises a nucleotide sequence which enables it to replicate within a bacterial cell is known under the name “BacMid”.
  • the replication-deficient baculovirus genome also comprises one or more nucleotide sequences encoding one or more selection markers making it possible to select or identify the bacterial cells transfected with the replication-deficient baculovirus genome.
  • These nucleotide sequences are not exogenous genes within the meaning of the invention.
  • selectable markers are ampicillin resistance gene, kanamycin resistance gene, hygromycin resistance gene, zeocin resistance gene and/or tetracycline resistance gene .
  • the replication-deficient baculovirus genome is obtained from a baculovirus genome selected from or derived from the genome of BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV or TeNPV, preferably AcMNPV.
  • the replication-defective baculovirus genome is in circular form.
  • the method according to the invention does not require linearization of the replication-defective genome.
  • the term “recombinant baculovirus genome” is intended to designate the baculovirus genome resulting from the recombination homolog between the baculovirus genome deficient for viral replication and the n transfer vectors.
  • the expression “recombinant baculovirus” is intended to designate a baculovirus whose genome is a recombinant baculovirus genome, that is to say a baculovirus encoding the n exogenous genes.
  • the recombinant baculovirus is produced after replication of the recombinant baculovirus genome in the insect cell.
  • the recombinant baculovirus can be secreted and can infect another insect cell.
  • the recombinant baculovirus of the invention is infectious.
  • gene is meant a nucleotide sequence capable of being transcribed into a protein or into a peptide of interest.
  • exogenous gene means a gene which is not naturally present in the genome of a baculovirus. They may be, for example, human genes, genes of animal origin, viral genes or bacterial genes. Moreover, an “exogenous gene” within the meaning of the invention is not present in the genome of baculovirus deficient for viral replication. Thus, the genes which are not naturally present in the genome of a baculovirus but which are present in the genome of baculovirus deficient for viral replication are not considered to be exogenous genes within the meaning of the present invention. In other words, the exogenous genes are carried by the transfer vectors which recombine with the baculovirus genome deficient for viral replication and which then find themselves integrated into the recombinant baculovirus.
  • an exogenous gene within the meaning of the present invention is placed under the control of appropriate elements for its expression in the insect cell.
  • appropriate elements is meant all the elements necessary for its transcription into messenger RNA (mRNA) and for the translation of the mRNA into protein.
  • the promoter is of particular importance. It may be a constitutive promoter or a regulatable promoter and it may be of baculoviral origin or of arthropod origin (eg of insect origin). What is important is that the chosen promoter is suitable for expression of the exogenous gene in the insect cell.
  • a promoter in use in the present invention can be modified so as to contain regulatory sequences.
  • promoters mention may be made of the polyhedrin promoter, the P10 promoter, the synthetic promoters derived from the polyhedrin and P10 promoters, the IE1 promoter of the baculovirus CfMNPV, the IE1 promoter of the baculovirus LdMNPV, the gp64 promoter of the baculovirus OpMNPV, the IE1 promoter of the shrimp virus WSSV (White spot syndrome virus), the P9 promoter of the densovirus of Junonia coenia (JcDNV), the A3 (actin 3) cell promoter of the silkworm Bombyx mori.
  • the polyhedrin promoter the P10 promoter
  • the synthetic promoters derived from the polyhedrin and P10 promoters the IE1 promoter of the baculovirus CfMNPV
  • the IE1 promoter of the baculovirus LdMNPV the gp64 promoter of the baculovirus OpMNPV
  • one or more of the exogenous genes is placed under the control of a synthetic promoter derived from the wild-type P10 promoter (SEQ ID NO: 1), preferably the synthetic P10S1A promoter (SEQ ID NO: 2) or P10S1B (SEQ ID NO:3).
  • expression cassette is meant a nucleotide sequence generally consisting of one or more genes and the elements suitable for its/their expression, for example an exogenous gene and the elements suitable for its expression in the insect cell.
  • the recombinant baculovirus makes it possible to express (i.e. to produce) the n exogenous genes in an insect cell.
  • the insect cell is chosen from Sf9, Sf21, Tn5-b14, Lepidopteran cell lines sensitive to the AcMNPV baculovirus or Sf21 lines, preferably Sf9.
  • Transfer vectors can also contain one or more nucleotide sequences which allow them to replicate within a bacterial cell. They can also contain genes encoding a selection marker making it possible to select or identify the bacterial cells transformed with a transfer vector.
  • One of the main advantages of the present invention is that the ability to replicate of the viral replication-deficient baculovirus genome is restored by recombination with the n transfer vectors.
  • each of the n transfer vectors encodes, in addition to one of the n exogenous genes, a nucleotide sequence making it possible to restore the function of one of the n non-functional genes essential to viral replication.
  • only recombination with the n transfer vectors is able to restore replication of the replication-deficient baculovirus genome.
  • the process of the invention guarantees that only the genomes of recombinant baculoviruses containing the n exogenous genes will be able to generate infectious recombinant baculoviruses. This method avoids having to use time-consuming tests to identify the recombinant baculoviruses containing the n exogenous genes.
  • the genes essential for viral replication are selected from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 ( ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89) ), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 ( ORF144), ORF146, ie1 (ORF147), lef-2 (ORF6).
  • the n non-functional genes essential for viral replication are each adjacent to a gene non-essential for viral replication.
  • the n exogenous genes each recombine at the level of a non-essential gene for viral replication adjacent to a non-functional gene essential for viral replication. Since the non-essential gene for viral replication is not essential for replication of the baculovirus genome, this recombination does not affect the ability of the baculovirus genome to replicate.
  • the expression “replication” or “viral replication” means the replication of both the baculovirus genome and the baculovirus. It being understood that the replication of the baculovirus genome in an insect cell is essential for the replication of the baculovirus in the insect cell. Thus, a gene essential for viral replication is understood as a gene essential for baculovirus replication.
  • the replication of baculovirus in the insect cell allows the generation of infectious baculovirus.
  • the method of the invention makes it possible to generate an infectious recombinant baculovirus in the insect cell.
  • a gene essential to non-functional viral replication is adjacent to a gene non-essential to viral replication when the two genes follow one another or partially overlap on the genome of the baculovirus, preferably no other gene is between the non-functional gene essential for viral replication and the non-essential gene for viral replication.
  • the two aforementioned genes are separated by an intervening nucleotide sequence, for example a non-coding intervening nucleotide sequence.
  • the spacer nucleotide sequence has a length ranging from 1 bp to 600 bp. It is also possible that no intervening nucleotide sequence (i.e.
  • the spacer nucleotide sequence may comprise a gene that is not essential for viral replication.
  • the choice of non-functional genes essential for viral replication adjacent to genes non-essential for viral replication was particularly advantageous and made it possible to obtain homogeneous homologous recombinations and therefore to prepare baculoviruses recombinants whose genomes are homogeneous.
  • only perfect recombination of the n vectors makes it possible to obtain a recombinant baculovirus capable of replicating in an insect cell.
  • the genomes of the recombinant baculoviruses prepared according to the invention be more than 90% homogeneous, advantageously more than 95%, preferably more than 99% and most preferably around 100%, preferably identical.
  • the non-essential gene for viral replication is chosen from Ph (ORF 8), ORF11, ORF13, egt (ORF15), u-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 ( ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37(ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif4 (ORF96) he65 (ORF105), ORF108, ORF110, cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), ORF5.
  • Ph ORF 8
  • the gene pair essential for viral replication/gene non-essential for viral replication is chosen from the pairs listed in the following table:
  • the expression “homologous recombination” is intended to denote the exchange of genetic information between two different nucleotide sequences, requiring the presence of homologous sequences between the two different nucleotide sequences.
  • the recombination takes place in the insect cell between (a) a replication-deficient baculovirus genome in which n genes essential for viral replication are non-functional and (b) the n vectors of transfer. Recombination in the context of the present invention takes place in a single step in the insect cell. That is, the recombination of the n exogenous genes with the replication-deficient baculovirus genome occurs simultaneously or nearly simultaneously in the insect cell.
  • the method according to the invention implements an intermolecular homologous recombination mechanism.
  • the homologous recombination mechanism consists of the exchange of homologous nucleotide sequences between the replication-deficient baculovirus genome and the n transfer vectors. These nucleotide sequences can be identical or substantially homologous.
  • the transfer vectors comprise, on either side of the exogenous gene expression cassette, flanking sequences homologous to the replication-deficient baculovirus genome. The degree of homology of the flanking sequences with the corresponding part of the replication-defective baculovirus genome can be variable but must be sufficient to allow intermolecular recombination.
  • flanking sequences can range from 10 bp (ie 10 base pairs) to 10 kb (ie 10,000 base pairs), advantageously ranging from 100 bp to 6 kb, preferably ranging from 200 bp to 6 kb and most preferably ranging from 400 bp to 6 kb.
  • the genetic material located between the flanking sequences of the n transfer vectors replaces the genetic material located between the two sequences homologous to the flanking sequences of the replication-deficient baculovirus genome.
  • This intermolecular exchange makes it possible to generate an infectious recombinant baculovirus in the insect cell.
  • all of the nucleotide sequences i) ie the nucleotide sequences making it possible to restore the function of the n genes essential for non-functional viral replication
  • the n transfer vectors are capable of restoring the replication of the baculovirus genome deficient for replication.
  • intermolecular exchange makes it possible to restore the function of non-functional n genes essential to viral replication.
  • the restoration of the function of the n non-functional genes essential for viral replication occurs when homologous recombination occurs correctly.
  • the n transfer vectors each comprise a nucleotide sequence making it possible to restore the function of one of the n non-functional genes essential to viral replication.
  • a replication-deficient baculovirus genome in which two genes essential for viral replication are non-functional recombines with two transfer vectors which each comprise a nucleotide sequence making it possible to restore the function of one of the two non-functional genes essential for viral replication.
  • the recombination with the first transfer vector makes it possible to restore the function of a first gene essential to non-functional viral replication and the recombination with the second transfer vector makes it possible to restore the function of a second gene essential for non-functional viral replication.
  • recombination with the n transfer vectors, or multirecombination is necessary to restore replication of the replication-deficient baculovirus genome.
  • the inventors demonstrated that multirecombination could take place in a single step in the insect cell.
  • This is particularly advantageous since the replication-deficient baculovirus genome and the n transfer vectors can be introduced into the insect cell at the same time, i.e. the replication-deficient baculovirus genome and the transfer vectors Transfer vectors are introduced simultaneously into the insect cell, in other words the replication-defective baculovirus genome and the transfer vectors are introduced in a single step into the insect cell.
  • Multirecombination in a single step during the process according to the invention is therefore very easy to implement and makes it possible to obtain homogeneous recombinant baculovirus genomes, that is to say that the homology of the nucleotide sequence of each genome of the recombinant baculovirus produced is greater than 90%, preferably greater than 95%, preferably greater than 99%, preferably around 100%, preferably equal to 100%.
  • the method according to the invention makes it possible to prepare a recombinant baculovirus expressing (i.e. producing) n exogenous polypeptides.
  • the n exogenous polypeptides form a single protein or several proteins.
  • the term “polypeptide” means a chain of amino acids linked by peptide bonds.
  • a polypeptide can be a protein, a protein subunit, a protein fragment, or simply a chain of amino acids.
  • a polypeptide is made up of at least 10 amino acids.
  • the n exogenous polypeptides form several proteins. They may be proteins comprising a single polypeptide chain, several identical subunits or several distinct subunits. The number of proteins produced can be equal to or less than n.
  • the n exogenous polypeptides form a single protein.
  • the protein generally comprises n distinct subunits.
  • the method according to the invention is particularly advantageous for preparing a recombinant baculovirus expressing (i.e. producing) an exogenous protein comprising several distinct subunits, for example a protein having its function only when it comprises all its subunits.
  • the subunits are generally linked together by non-covalent bonds (e.g. hydrophobic bonds) and/or covalent bonds (e.g. disulphide bridges between two cysteines).
  • the method according to the invention makes it possible to easily and quickly prepare a recombinant baculovirus comprising n distinct exogenous genes which encode a single protein comprising n distinct subunits.
  • the term “distinct exogenous genes” means genes not having the same nucleotide sequence.
  • the term “distinct subunits” means peptide sequences not having the same amino acid sequence.
  • a "protein comprising n distinct subunits" comprises n distinct subunits linked together by non-covalent bonds and/or covalent bonds.
  • the number of transfer vectors will depend on the number of distinct subunits that the protein comprises.
  • each transfer vector comprises an exogenous gene different from the exogenous genes included in the other transfer vectors.
  • the recombinant baculovirus of the invention cannot comprise all of the protein of interest (that is to say all of the distinct subunits which constitute the protein of interest) only if the recombination takes place between the replication-deficient baculovirus genome and the transfer vectors.
  • n is an integer at least equal to 2, for example an integer ranging from 2 to 30, for example ranging from 2 to 10.
  • the value of n will correspond to the number of distinct subunits which constitute said protein.
  • n 2 and the recombinant baculovirus encodes 2 exogenous genes which encode a protein comprising two subunits, advantageously 2 distinct exogenous genes which encode a protein comprising 2 distinct subunits.
  • the protein is an antibody, a protein complex forming Virus-Like-Particles (VLP), a couple of viral proteins, a peptide hormone, preferably an antibody.
  • VLP protein complex forming Virus-Like-Particles
  • the protein is a bispecific antibody, a set of three viral proteins, a multi-enzyme complex, a protein complex, a VLP-forming protein complex, preferably a bispecific antibody.
  • antibody is used herein in the broadest sense and encompasses various antibody structures widely described in the literature, including but not limited to antibodies of any origin, monoclonal antibodies, polyclonal antibodies and antibody fragments as long as they exhibit the desired activity (eg antigen binding).
  • the antibody is an IgA, IgD, IgE, IgG or an IgM.
  • antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fc fragments; diabodies; scFv/Fc; camelid type antibodies (e.g. VHH); single chain antibody molecules (e.g. scFv).
  • the recombinant baculovirus prepared according to the method of the invention does not comprise a nucleic acid sequence which allows it to replicate within a bacterial cell.
  • the nucleic acid sequence which makes it possible to replicate the replication-deficient baculovirus genome within a bacterial cell is eliminated during homologous recombination in the insect cell.
  • the introduction into an insect cell of the transfer vectors and of the replication-deficient baculovirus genome is carried out according to the general techniques widely described in the prior art. Mention may in particular be made of the calcium phosphate technique, the DEAE dextran technique, electroporation, methods based on osmotic shock, microinjection or methods based on the use of liposomes, preferably lipofection.
  • the transfer vectors and the replication-deficient baculovirus genome are introduced at the same time into the insect cell, i.e. the transfer vectors and the baculovirus genome deficient for replication are introduced simultaneously into the insect cell, in other words the transfer vectors and the replication deficient baculovirus genome are introduced in a single step.
  • the amounts of replication-deficient baculovirus genome and of transfer vectors introduced into the insect cell can vary. It is preferred to employ a 5-fold larger amount of each of the transfer vectors relative to the amount of replication-deficient baculovirus genome.
  • Another advantage of the present invention is that the replication-deficient baculovirus genome is introduced in circular form, i.e. without a step of linearization beforehand. This linearization step is useless since the baculovirus genome is deficient for replication, even in circular form, since it comprises non-functional genes essential for viral replication.
  • the absence of a linearization step is another major advantage of the present invention.
  • the present description also relates to a recombinant baculovirus comprising (ie “recombinant baculovirus whose genome comprises” or “recombinant baculovirus whose genome encodes”) n nucleotide sequences of formula (I): embarrassed exogenous ⁇ sequence nucleotide interlayer ⁇ embarrassed essential at the replication viral functional said spacer nucleic acid sequence consists of 0 (zero) to 600 bp, said functional viral replication essential gene is selected from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef- 11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), Ief-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89),
  • the recombinant baculovirus does not comprise a nucleic acid sequence which allows it to replicate within a bacterial cell. It has in fact been demonstrated that the absence of such a sequence made it possible to increase the stability of the recombinant baculovirus, compared to a recombinant baculovirus comprising such a sequence (Pijlman et al (2003), Spontaneous excision of BAC vector sequences from bacmid -derived baculovirus expression vectors upon passage in insect cells. Journal of General Virology).
  • the recombinant baculovirus does not include n genes non-essential to viral replication chosen from Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-ubiquitin (ORF35), 39K (ORF36), ORF38, p43 ( ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37(ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 ( ORF96) he65 (ORF105), ORF108, ORF110, Cathepsin (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), ORF5.
  • one of the n genes non-essential to viral replication is the gene encoding cathe
  • n is an integer at least equal to 2, for example an integer ranging from 2 to 30, for example ranging from 2 to 10, as detailed in the “preparation of the recombinant baculovirus” section above.
  • the present description also relates to a recombinant baculovirus, which can be obtained according to the preparation process of the invention, comprising n nucleotide sequences of formula (I): embarrassed exogenous ⁇ sequence nucleotide interlayer ⁇ embarrassed essential at the replication viral functional said spacer nucleic acid sequence consists of 0 (zero) to 600 bp, said functional viral replication essential gene is selected from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef- 11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF
  • the n nucleotide sequences are distributed over the entire genome of the recombinant baculovirus, which makes it possible to improve its stability.
  • the n exogenous genes chosen are therefore sufficiently spaced out on the baculovirus genome. This characteristic is implicit since the distribution on the genome is linked to the “essential gene/non-essential gene” pairs.
  • nucleotide sequences of formula (I) are not duplicated on the genome of the recombinant baculovirus. It has in fact been demonstrated that the stability of the recombinant baculovirus is reduced when the sequences are duplicated on the genome (data not shown).
  • the non-functional genes essential for viral replication are each adjacent to a gene non-essential for viral replication, as described in the “preparation of the recombinant baculovirus” section above.
  • the transfer vectors comprise, on either side of the exogenous gene expression cassette, flanking sequences homologous to the replication-deficient baculovirus genome.
  • the flanking sequences of each of the transfer vectors are homologous to all or part of said non-functional viral replication essential gene and to all or part of said non-essential viral replication gene.
  • the flanking sequences have a length which can range from 10 bp (i.e. 10 base pairs) to 10 kb (i.e. 10,000 base pairs), advantageously ranging from 100 bp to 6 kb, preferably ranging from 200 bp to 6 kb and, most preferably ranging from 400 bp to 6 kb.
  • the flanking sequences are described in detail in the “preparation of the recombinant baculovirus” section above.
  • the n transfer vectors each comprise an exogenous gene.
  • the n exogenous genes encode n exogenous polypeptides.
  • the n exogenous polypeptides form a single protein or several proteins, as detailed in the “preparation of the recombinant baculovirus” part above.
  • n 2 and the 2 exogenous genes encode a protein comprising two subunits, advantageously 2 distinct exogenous genes which encode a protein comprising 2 distinct subunits.
  • the protein is a monospecific antibody, a VLP, a couple of viral proteins, a peptide hormone, preferably an antibody.
  • n 3 and the 3 exogenous genes encode a protein comprising three subunits, advantageously 3 distinct exogenous genes which encode a protein comprising 3 distinct subunits.
  • the protein is a bispecific antibody, a set of three viral proteins, a multi-enzyme complex, a protein complex, a VLP composed of 3 distinct proteins, preferably a bispecific antibody.
  • the replication-defective baculovirus genome is obtained from a baculovirus genome selected from or derived from the genome of BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV or TeNPV, preferably AcMNPV.
  • the present description also relates to a cell comprising a recombinant baculovirus or a cell comprising a set of homologous recombination elements.
  • the cell is an insect cell, preferably selected from Sf9, Sf21, Tn5-b14, lepidopteran cell lines sensitive to the AcMNPV baculovirus, Sf21 lines, preferably Sf9, as described further below. detail in the “preparation of the recombinant baculovirus” section above.
  • the genes essential for viral replication are chosen from 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), Ief-3 (ORF67), ORF73, ORF75, ORF81, p95 ( ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), helicase (ORF95), vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), lef-2 (ORF6), as detailed above in the “pre
  • the n non-functional essential genes for viral replication are each adjacent to a non-essential gene for viral replication, as detailed in the “preparation of the recombinant baculovirus” section below. above.
  • the gene non-essential to viral replication is selected from those detailed above in the “preparation of the recombinant baculovirus” part.
  • the n exogenous genes each recombine at the level of a non-essential gene for viral replication adjacent to a non-functional gene essential for viral replication, as detailed in the “preparation of the recombinant baculovirus” section above.
  • the transfer vectors each comprise, on either side of the exogenous gene expression cassette, flanking sequences homologous to the replication-deficient baculovirus genome.
  • the flanking sequences of each of the transfer vectors are homologous to all or part of said non-functional viral replication-essential gene and to all or part of said non-essential viral replication gene.
  • the flanking sequences have a length which can range from 10 bp (i.e. 10 base pairs) to 10 kb (i.e. 10,000 base pairs), advantageously ranging from 100 bp to 6 kb, preferably ranging from 200 bp to 6 kb and, most preferably ranging from 400 bp to 6 kb.
  • the flanking sequences are described in detail in the “preparation of the recombinant baculovirus” section above.
  • the replication-deficient baculovirus genome is obtained from a baculovirus genome selected from or derived from the baculovirus genome BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV , MbMNPV, OpMNPV, SIMNPV, SeMNPV or TeNPV, preferably AcMNPV., as detailed in the “preparation of the recombinant baculovirus” section above.
  • the n exogenous polypeptides produced by the method described form several proteins. They may be proteins comprising a single polypeptide chain or several distinct subunits. The number of proteins produced can be equal to or less than n.
  • the n exogenous polypeptides produced by the method described form a single protein.
  • the protein generally comprises n distinct subunits.
  • the protein is a monoclonal antibody, a VLP composed of 3 distinct proteins, a couple of viral proteins, a peptide hormone, a bispecific antibody, a set of three viral proteins, a multienzymatic complex, a protein complex.
  • the insect cell is selected from Sf9, Sf21, Tn5-b14, lepidopteran cell lines sensitive to the AcMNPV baculovirus, the Sf21 lines, preferably Sf9, as detailed in the "preparation of the baculovirus recombinant” above.
  • the replication-deficient baculovirus genome is obtained from a baculovirus genome selected from or derived from the baculovirus genome BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV , MbMNPV, OpMNPV, SIMNPV, SeMNPV or TeNPV, preferably AcMNPV, as detailed in the “preparation of the recombinant baculovirus” section above.
  • the replication-deficient baculovirus genome comprises at least one nucleotide sequence which allows it to replicate within a bacterial cell, as detailed in the “preparation of the recombinant baculovirus” section above.
  • the recombinant baculovirus capable of replication does not comprise a nucleotide sequence which allows it to replicate within a bacterial cell, as detailed in the “preparation of the recombinant baculovirus” section above.
  • condition suitable for producing the exogenous polypeptides is understood to mean the conditions under which the insect cells are cultured to produce the exogenous polypeptides, in particular a culture medium suitable for the optimal growth of the cells, an optimal temperature and an optimal pH.
  • the culture medium may contain a serum of animal origin.
  • the production method described further comprises a step b′) introducing the replication-deficient baculovirus genome and the n transfer vectors into an insect cell.
  • the introduction of the vectors into an insect cell is carried out according to the general techniques widely described in the prior art. Mention may in particular be made of the calcium phosphate technique, the DEAE dextran technique, electroporation, methods based on osmotic shock, microinjection or methods based on the use of liposomes, preferably lipofection.
  • the replication-deficient baculovirus genome and the transfer vectors are introduced at the same time into the insect cell, that is to say simultaneously into the insect cell, in d
  • the replication-defective baculovirus genome and transfer vectors are introduced into the insect cell in a single step.
  • the replication-deficient baculovirus genome is introduced into the insect cell in circular form, ie it is not linearized beforehand. As detailed above, the introduction in circular form is another major advantage of the production process described.
  • the present description also relates to the use of a recombinant baculovirus obtained according to the process according to the invention or of a cell as described for the production of n exogenous polypeptides encoded by n exogenous genes.
  • Example 1 Construction of a replication-deficient baculovirus genome in which 1 gene essential for viral replication is non-functional (BacMid 1)
  • BacMid 1 presents the deletion of a gene essential for viral replication, gene 1629.
  • This operation is carried out in the insect cell.
  • the Mini-F bacterial origin of replication was introduced into the polyhedrin locus of the AcMNPV baculovirus genome by homologous recombination in Sf9 ( Spodoptera frugiperda ) insect cells.
  • Sf9 Spodoptera frugiperda
  • the cells were transfected with (i) a PH transfer vector (pVT/Mini-F Kan R ) in which the sequence of the ph gene was replaced by a DNA fragment carrying the Mini-F + a cassette of bacterial expression conferring resistance to Kanamycin (Kan R ), and (ii) an AcMNPV baculovirus genome (Baculovirus isolated from the lepidopteran Autographa californica).
  • the generated baculoviruses were purified by the plaque lysis technique and then characterized in order to confirm that they had indeed integrated the Mini-F and the Kan R expression cassette.
  • a baculovirus was selected and was then transferred into the bacterium E. coli EL350, thus generating a first BacMid (BacMid 0, not deficient for viral replication in insect cells).
  • Kan R bacterial expression by homologous recombination in E. coli EL350 bacteria . During this recombination, a DNA fragment encoding the 27 C-terminal amino acids of the 1629 protein was deleted, rendering the 1629 protein non-functional. The ampicillin resistance gene was then eliminated after digestion with MauBI then religation, thus generating BacMid 1.
  • BacMid 1 The baculovirus genome (ie BacMid 1) is then deficient for replication in insect cells, because a gene essential for viral replication (ie the gene encoding protein 1629) is non-functional.
  • the bacteria containing BacMid 1 are hereinafter referred to as “ E. coli EL350/BacMid1 bacteria”.
  • the figure 1 illustrates the preparation steps for BacMid 1.
  • Example 2 Construction of a replication-deficient baculovirus genome in which 2 genes essential for viral replication are non-functional (BacMid 2)
  • BacMid 2 presents the deletion of 2 genes essential for viral replication, gene 1629 and the gene encoding viral DNA polymerase (DNAPol). From BacMid 1, deletion of the DNAPol gene was carried out in E. coli EL350/BacMid1 bacteria after electroporation of a 4222 bp recombination fragment in which part of the genes encoding gp37 (240 amino acids) and DNAPol (466 C-terminal amino acids) has been deleted and replaced by a bacterial expression cassette allowing the production of hygromycin B phosphotransferase (Hygro R ) thus conferring resistance to hygromycin (Hygro R ).
  • Hygro R hygromycin B phosphotransferase
  • the Hygro R gene was placed under the control of the bacterial promoter EM7 (from the commercial vector pSelect-Hygro-mcs, Invitrogen), the glms terminator was introduced downstream of the Hygro R gene ( Gay NJ et al. Biochem J., 1986, 234, 111-117 ).
  • the bacteria containing BacMid2 E. coli EL350/BacMid2 were selected for their resistance to hygromycin.
  • the baculovirus genome ie BacMid 2 is deficient for replication in insect cells, since two genes essential for viral replication (ie the gene encoding the 1689 protein and the gene encoding DNAPol) are non-functional.
  • the figure 2 is a diagram that illustrates the DNAPol deletion step for the preparation of BacMid 2.
  • BacMid 2 can be used to produce a single protein (see example 4). It suffices to have two transfer vectors, one bringing the exogenous gene to be produced and all or part of the deleted gene 1 and the other bringing the wild-type gene corresponding to the deleted gene 2. The two deleted genes are repaired during the homologous recombination.
  • Example 3 Construction of a replication-deficient baculovirus genome in which 3 genes essential for viral replication are non-functional (BacMid 3)
  • BacMid 3 presents the deletion of 3 essential genes, 1629, DNAPol and gp64.
  • the deletion of the gp64 gene was carried out in E. coli EL350/BacMid2 bacteria after electroporation of a 3260bp recombination fragment in which the entire cathepsin gene plus 779 bp of the coding sequence 259 aa of chitinase and part of the gp64 gene, deletion of 566 bp encoding 188 amino acids, a been replaced by a bacterial expression cassette conferring resistance to Zeocin ( Drocourt et al., Nucleic Acids Research, vol.18n°13, 1990 ).
  • the Zeocin ® gene from the commercial plasmid pCR ® -Blunt (Invitrogen) was placed under the control of the bacterial promoter T5N25, from phage T5 ( Gentz and Bujard, J. Bacteriology, vol.164n°1, 1985 ) and tracking the transcription terminator rrnBT1 ( E.coli ribosomal RNA operon T1 terminator) ( Kwon et al., J Biol. Chem., vol 274 n°41, 1999 ).
  • Bacteria containing BacMid3 E. coli EL350/BacMid3 were selected for their resistance to Zeocin.
  • the baculovirus genome (ie BacMid 3) is deficient for replication in insect cells, because three genes essential for viral replication (ie the gene encoding protein 1689 and the gene encoding DNAPol and the gene encoding gp64) are not -functional.
  • the picture 3 is a diagram that illustrates the gp64 deletion step for the preparation of BacMid 3.
  • a transfer vector pVT/gp37 was constructed in order to be able to generate recombinant baculoviruses expressing 2 exogenous genes.
  • the EcoRI F fragment of the AcMNPV baculovirus genome containing the gp37 gene and the DNAPol gene was cloned into a bacterial plasmid pUC, thus generating the pUC/gp37.
  • This plasmid was then modified as follows: a large part of the gene encoding gp37 was deleted (724 bp), the initiator ATG was mutated and replaced by two unique restriction sites XbaI and Avril allowing the integration of an exogenous gene under the control of the natural gp37 promoter. These modifications thus led to the production of the transfer vector pVT/gp37.
  • Sf9 cells were transfected by lipofection with pVT/PH and pVT/gp37 transfer vectors loaded with exogenous genes and BacMid 2 DNA. Viruses generated after homologous recombination were cloned by the plaque lysis method. . The production of the recombinant protein was verified by an appropriate method (e.g. for example ELISA, Western blot, enzymatic assay). The genome of the recombinant viruses was verified by Southern blot and the sequence of the exogenous integrated into the viral genome was verified by sequencing after PCR amplification.
  • an appropriate method e.g. for example ELISA, Western blot, enzymatic assay
  • the figure 4 is a diagram which illustrates the transfer vector pVT/gp37 for the expression of a gene X (where X is a gene different from the gene encoding the heavy chain of an antibody).
  • the recombinant baculovirus genomes generated after homologous recombination between BacMid2 and the transfer vectors no longer express gp37 (non-essential protein for viral replication).
  • pVT/PH containing a wild-type sequence, that is to say containing the wild-type (unmodified) expression cassette leading to the production of polyhedrin.
  • the pVT/PH could also be “empty”, that is to say not contain any exogenous gene or the polyhedrin gene.
  • the figure 5 is a diagram which illustrates the construction and use of the PH transfer vector pVT/PH for the expression of an exogenous gene X (where X is an exogenous gene different from the gene encoding an antibody light chain).
  • the figure 6 is a diagram which illustrates the construction and use of the transfer vector pVT/gp37C ⁇ 1 for the expression of the heavy chain of an antibody.
  • the figure 7 is a diagram which illustrates the construction and use of the transfer vector pVT/PHC• for the expression of the light chain of an antibody.
  • the BstXI-XbaI fragment from the EcoRI E and H regions of the AcMNPV baculovirus was cloned into a pUC plasmid.
  • An EcoNI-EcoRI deletion of 1175 bp makes it possible to inactivate the genes encoding chitinase ( chiA ), non-essential in vitro, and also non-essential cathepsin ( v-cath ).
  • the addition of an XbaI site between the EcoNI and EcoRI sites makes it possible to integrate an exogenous gene.
  • the Sf9 cells are transfected by lipofection with the transfer vectors pVT/PH, pVT/gp37 and pVT/chitCath loaded with the exogenous genes and the BacMid 3 DNA.
  • the viruses generated during the homologous recombination were cloned by the lysis plaque method.
  • the production of the recombinant protein was checked by an appropriate method, ELISA, Western blot, enzymatic assay... the genome of the recombinant viruses was checked by Southern blot and the sequence of the exogenous gene was checked after PCR amplification.
  • the figure 8 is a diagram which illustrates the construction of the pVT/Chit-Cath vector and its homologous recombination with BacMid 3.
  • Example 6 Production of an anti-CD4 monoclonal antibody (13B8II) using BacMid 2.
  • the Sf9 cells were transfected by lipofection with BacMid 2 and the 2 transfer vectors obtained in Example 4 and then incubated for 4 days at 28°C.
  • the culture supernatants were removed and the generated recombinant baculoviruses, secreted into the culture medium, were cloned by the lysis plaque technique.
  • Example 7 Use of BacMid 3 for the production of VLP (Virus-Like-Particle).
  • the Sf9 cells were transfected by lipofection with BacMid 3 and the 3 transfer vectors obtained above, then incubated for 4 days at 28°C.
  • the recombinant baculoviruses generated and then secreted into the culture supernatant were cloned by the lysis plaque method.
  • the organization of the recombinant baculovirus genomes was checked by Southern blot (see figure 10 ) and the integrated genes were verified after PCR amplification, cloning and then sequencing.
  • the Southern blot was carried out on the genomic DNA of the recombinant virus expressing the 3 proteins HA, NA and M of the influenza virus to detect the cDNA encoding the integrated M, HA and NA proteins.
  • the membranes were hybridized with probes specific for these 3 genes.
  • Example 8 Use of BacMid 3 for the production of bispecific antibodies.
  • the bispecific antibody constructed according to the patent (PCT/IB2012/053482) consists of a heavy chain composed of the VH+CH1+CH2+CH3 domains of an antibody 1, fused at the N-terminal to the VH+CH1 domains of an antibody 2. Mutations introduced at the interface of the CL and CH1 regions of antibody 1 promote correct pairings between the VL1 and VL2 domains of the L1 and L2 light chains which are produced separately and the corresponding VH1 and VH2 domains.
  • the production of this antibody requires the simultaneous production and in equal quantity of 3 chains, the fused heavy chain, the L1 light chain and the L2 light chain.
  • the cDNA encoding the L1 light chain was introduced into the transfer vector pVT/PH as described in figure 5 .
  • the cDNA encoding the L2 light chain was introduced into the transfer vector pVT/gp37 as described in figure 4 .
  • the cDNA encoding the fused heavy chain was introduced into the pVT/Chit-Cath transfer vector as described in figure 8 .
  • the figure 11 is a diagram which illustrates in A the structure of the bispecific antibody in B the analysis by polyacrylamide gel electrophoresis of the bispecific antibody purified on a column of protein A Sepharose.

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Claims (10)

  1. Verfahren zur Herstellung, in einer Insektenzelle, eines rekombinanten Baculovirus, umfassend n interessierende Gene, durch homologe Multirekombination zwischen:
    a) einem für die Replikation defizienten Baculovirusgenom, wobei für die virale Replikation n wesentliche Gene nichtfunktional sind; und
    b) n Transfervektoren, die jeweils umfassen:
    i) eine Nukleotidsequenz, die erlaubt, die Funktion eines der für die virale Replikation n wesentlichen nichtfunktionalen Gene wiederherzustellen,
    ii) eins der n interessierende Gene,
    wobei die Gesamtheit der Nukleotidsequenzen i) der n Transfervektoren imstande ist, die Replikation des für die Replikation defizienten Baculovirusgenoms wiederherzustellen;
    wobei n eine Ganzzahl von mindestens gleich 2 ist, und
    das für die Replikation defiziente Bakulovirusgenom und die n Transfervektoren in einem einzigen Schritt in die Insektenzelle eingeführt werden.
  2. Verfahren nach Anspruch 1, wobei die für die virale Replikation wesentlichen Gene aus 1629 (ORF9), Pk1 (ORF10), lef-1 (ORF14), ORF34, lef-11 (ORF37), p47 (ORF40), lef8 (ORF50), DNAJ Domain (ORF51), ORF53, vp1054 (ORF54), Lef-9 (ORF62), DNA Pol (ORF65), lef-3 (ORF67), ORF73, ORF75, ORF81, p95 (ORF83), vp39 (ORF89), lef-4 (ORF90), p33 (ORF92), Helicase (ORF95), vp80 (ORF104), ORF106-107, odv-ec43 (ORF109), gp64/67 (ORF128), ORF132, ORF133, odv-ec27 (ORF144), ORF146, ie1 (ORF147), lef-2 (ORF6) ausgewählt sind.
  3. Verfahren nach einem der Ansprüche 1 oder 2, wobei die für virale Replikation n wesentlichen nichtfunktionalen Gene jeweils an ein für die virale Replikation nichtwesentliches Gen angrenzen.
  4. Verfahren nach Anspruch 3, wobei das für die virale Replikation nichtwesentliche Gen aus Ph (ORF 8), ORF11, ORF13, egt (ORF15), v-Ubiquitin (ORF35), 39K (ORF36), ORF38, p43 (ORF39), lef-12 (ORF41), pcna (ORF49), ORF52, ORF55, Fp (ORF61), ORF63, gp37 (ORF64), ORF68, ORF72, ORF74, ORF82, cg30 (ORF88), ORF91, pif-4 (ORF96), he65 (ORF105), ORF108, ORF110, Cathepsine (ORF127), p24 (ORF129), pp34 (ORF131), ORF134, ORF145, odv-e56 (ORF148), ORF5 ausgewählt ist.
  5. Verfahren nach einem der Ansprüche 3 oder 4, wobei die n interessierenden Gene jeweils im Bereich eines für die virale Replikation nichtwesentlichen Gens rekombinieren, das an ein für die virale Replikation wesentliches nichtfunktionales Gen angrenzt.
  6. Verfahren nach einem der Ansprüche 1 bis 5, wobei die Transfervektoren beiderseits der Expressionskassette des interessierenden Gens flankierende Sequenzen umfassen, die zum für die Replikation defizienten Bakulovirusgenom homolog sind.
  7. Verfahren nach Anspruch 6, wobei die flankierenden Sequenzen eine Länge haben, die von 10 pb (d. h. 10 Basenpaare) bis 10 kb (d. h. 10.000 Basenpaare), in vorteilhafter Weise von 100 pb bis 6 kb, vorzugsweise von 200 pb bis 6 kb und sehr bevorzugt von 400 pb bis 6 kb reicht.
  8. Verfahren nach einem der Ansprüche 1 bis 7, wobei die Insektenzelle aus Sf9, Sf21, Tn5-b14, den für Baculovirus sensiblen Schmetterlings-Zelllinien AcMNPV, den Linien Sf21, vorzugsweise Sf9, ausgewählt ist.
  9. Verfahren nach einem der Ansprüche 1 bis 8, wobei das für die Replikation defiziente Bakulovirusgenom aus einem Bakulovirusgenom gewonnen wird, das aus oder von dem Genom von BmNPV, AcMNPV, ApNPV, BsSNPV, CfMNPV, EoSNPV, HaNPV, HzNPV, LdMNPV, MbMNPV, OpMNPV, SIMNPV, SeMNPV oder TeNPV, vorzugsweise AcMNPV, ausgewählt oder abgeleitet ist.
  10. Verfahren nach einem der Ansprüche 1 bis 9, wobei n eine Ganzzahl von 2 bis 30, beispielsweise von 2 bis 10 ist.
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EP3494127A1 (de) 2019-06-12
FR3054841B1 (fr) 2021-01-29

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